Trimethoxyphenyl Ring Research Articles

Abstract 2676: Unusual binding of TMP in two different sites in the binary complex with the Q35S/N64F double mutant of human dihydrofolate reductase

Abstract In an effort to understand the role of specific active site residues in modulating the binding selectivity of inhibitors against dihydrofolate reductase (DHFR) from different species, a series of single and double mutants of DHFR were made at positions 35 and 64 of human (h) DHFR. To explore sequence differences between human and the pathogenic organisms Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) DHFR, the causative agent of Pneumocystis pneumonia (PcP) in patients with AIDS, kinetic data were measured for trimethoprim, used for treatment of AIDS-related pneumonia. Crystal structure data are reported for the Q35S/N64F double mutant of hDHFR as a binary complex with trimethoprim (TMP). These substitutions make the human enzyme resemble a cross between pjDHFR which has Ser/Ser at these positions and pcDHFR which has Lys/Phe at these sites. Kinetic data show enhanced affinity for the binding of TMP to this double mutant (Ki 604 nM) compared to wild type hDHFR (Ki 1757 nM). The Ki of TMP is 43 nM for pjDHFR and 222 nM for pcDHFR. Structural data for the binary complex of TMP with this double mutant of hDHFR revealed the unexpected binding of two copies of TMP; one bound in the normal binding site while the other occupies a shallow pocket different from that involved in binding the γ-glutamate of folate substrates or inhibitors. The trimethoxyphenyl ring of the second TMP makes a series of hydrophobic contacts with the trimethoxyphenyl ring of the first TMP and with the phenyl ring of residue F31 and the mutated F64 in this structure. Structural data for the binary complex of wild type hDHFR with TMP does not indicate such binding. The conformation of the two TMP in the double mutant complex differ significantly from each other and from that observed for the wild type hDHFR binary complex. Analysis of all DHFR TMP complexes reveals the bridge torsion angles fall into two broad ranges and the two TMP in the mutant hDHFR complex fall into the less populated group. These data suggest that the combination of hydrophobic contacts from the normal F31 in hDFHR and the mutant F64 provide a unique environment to create sufficient van der Waals contact energy to stabilize binding in this unusual site. Supported in part by NIH GM51670 (VC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2676.

6-(4-Pyridyl)-3-(3,4,5-trimethoxyphenyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazole

In the mol­ecule of the title compound, C17H15N5O3S, the planar central heterocylic ring system is oriented at dihedral angles of 5.32 (4) and 9.41 (4)°, respectively with respect to trimethoxy­phenyl and pyridine rings. Intra­molecular C—H⋯N, C—H⋯O and C—H⋯S hydrogen bonds result in the formation of a nearly planar six-membered ring, which is oriented at a dihedral angle of 3.07 (5)° with respect to the central heterocylic ring system, and non-planar six- and five-membered rings having twist and envelope conformations, respectively. In the crystal structure, inter­molecular C—H⋯N and C—H⋯O hydrogen bonds link the mol­ecules. There is a C—H⋯π contact between the pyridine ring and a methyl group and a π–π contact between the central heterocylic ring system and the trimethoxy­phenyl ring [centroid–centroid distance = 3.758 (1) Å].

3,6-Bis(3,4,5-trimethoxyphenyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazole

In the mol­ecule of the title compound, C21H22N4O6S, the planar central heterocyclic ring system is oriented with respect to the trimethoxy­phenyl rings at dihedral angles of 2.60 (5) and 3.60 (6)°. Intra­molecular C—H⋯N and C—H⋯S hydrogen bonds result in the formation of planar five- and six-membered rings. In the crystal structure, inter­molecular C—H⋯O hydrogen bonds link the mol­ecules. There is a C—H⋯π contact between a methyl group and a trimethoxy­phenyl ring, and a π–π contact between the central heterocyclic ring system and a trimethoxy­phenyl ring [centroid–centroid distance = 3.640 (1) Å].

4-(2-Methoxyphenyl)-3-(3,4,5-trimethoxyphenethyl)-2H-1,2,4-triazole-5(4H)-thione

The title compound, C20H23N3O4S, is an important biologically active heterocyclic compound. The five-membered ring is oriented with respect to the six-membered rings at dihedral angles of 78.60 (3) (trimethoxyphenyl ring) and 71.57 (3)° (methoxyphenyl ring). In the crystal structure, inter­molecular N—H⋯O hydrogen bonds link the mol­ecules into infinite chains along the c axis.

Synthesis and biological activity of naphthalene analogues of phenstatins: Naphthylphenstatins

Novel phenstatin analogues with a 2-naphthyl moiety combined with either a 2,3,4- or a 3,4,5-trimethoxyphenyl ring have been synthesized, and their tubulin polymerization inhibiting and cytotoxic activities have been evaluated. The 2-naphthyl ring is a better replacement for the 3-hydroxy-4-methoxyphenyl ring in the phenstatin series than in the combretastatin series. For the naphthylphenstatins, the carbonyl is required, and the preferred orientation of the trimethoxyphenyl ring is the one found in combretastatins.

Highly Efficient Ligands for Dihydrofolate Reductase from Cryptosporidium hominis and Toxoplasma gondii Inspired by Structural Analysis

The search for effective therapeutics for cryptosporidiosis and toxoplasmosis has led to the discovery of novel inhibitors of dihydrofolate reductase (DHFR) that possess high ligand efficiency: compounds with high potency and low molecular weight. Detailed analysis of the crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis and a homology model of DHFR from Toxoplasma gondii inspired the synthesis of a new series of compounds with a propargyl-based linker between a substituted 2,4-diaminopyrimidine and a trimethoxyphenyl ring. An enantiomerically pure compound in this series exhibits IC50 values of 38 and 1 nM against C. hominis and T. gondii DHFR, respectively. Improvements of 368-fold or 5714-fold (C. hominis and T. gondii) relative to trimethoprim were generated by synthesizing just 14 new analogues and by adding only a total of 52 Da to the mass of the parent compound, creating an efficient ligand as an excellent candidate for further study.

Colchitaxel, a coupled compound made from microtubule inhibitors colchicine and paclitaxel.

BackgroundTumor promoters enhance tumor yield in experimental animals without directly affecting the DNA of the cell. Promoters may play a role in the development of cancer, as humans are exposed to them in the environment. In work based on computer-assisted microscopy and sophisticated classification methods, we showed that cells could be classified by reference to a database of known normal and cancerous cell phenotypes. Promoters caused loss of properties specific to normal cells and gain of properties of cancer cells. Other compounds, including colchicine, had a similar effect. Colchicine given together with paclitaxel, however, caused cells to adopt properties of normal cells. This provided a rationale for tests of microtubule inhibitor combinations in cancer patients. The combination of a depolymerizing and a stabilizing agent is a superior anti-tumor treatment. The biological basis of the effect is not understood.ResultsA single compound containing both colchicine and paclitaxel structures was synthesized. Colchicine is an alkaloid with a trimethoxyphenyl ring (ring A), a ring with an acetamide linkage (ring B), and a tropolone ring (ring C). Although rings A and C are important for tubulin-binding activity, the acetamide linkage on ring B could be replaced by an amide containing a glutamate linker. Alteration of the C-7 site on paclitaxel similarly had little or no inhibitory effect on its biological activity. The linker was attached to this position. The coupled compound, colchitaxel (1), had some of the same effects on microtubules as the combination of starting compounds. It also caused shortening and fragmentation of the + end protein cap.ConclusionSince microtubule inhibitor combinations give results unlike those obtained with either inhibitor alone, it is important to determine how such combinations affect cell shape and growth. Colchitaxel shows a subset of the effects of the inhibitor combination. Thus, it may be able to bind the relevant cellular target of the combination. It will be useful to determine the basis of the shape reversal effect and possibly, the reasons for therapeutic efficacy of microtubule inhibitor combinations.

Synthesis and anti-tubulin evaluation of chromone-based analogues of combretastatins

Twenty new hybrid compounds with both combretastatin and flavone moieties were synthesized. These derivatives are classified according to the position of the trimethoxyphenyl ring at C-2 or C-3 of the chromone and presence or absence of a carbonyl as a linker between C-3 and the aryl ring. Most of these compounds were prepared from hesperidin or naringin, two natural and abundant Citrus flavonoids. Seven of these combretastatin analogues revealed anti-tubulin activity but in a medium range.

Structural concept for fluorinated Y-enynes with solvatochromic properties.

An approach to the development of fluorescent probes to follow polymerizations in situ using fluorinated cross-conjugated enediynes (Y-enynes) is reported. Different substitution patterns in the Y-enynes result in distinct solvatochromic behavior. beta,beta-Bis(phenylethynyl)pentafluorostyrene 7, which bears no donor substituents and only fluorine at the styrene moiety, shows no solvatochromism. Donor substituted beta,beta-bis(3,4,5-trimethoxyphenylethynyl)pentafluorostyrene 8 and beta,beta-bis(4-butyl-2,3,5,6-tetrafluorophenylethynyl)-3,4,5-trimethoxystyrene 9 exhibit solvatochromism upon change of solvent polarity. Y-enyne 8 showed the largest solvatochromic shift (94 nm bathochromic shift) upon changing solvent from cyclohexane to acetonitrile. A smaller solvatochromic response (44 nm bathochromic shift) was observed for 9. Lippert-Mataga treatment of 8 and 9 yields slopes of -10,800 and -6,400 cm(-1), respectively. This corresponds to a change in dipole moment of 9.6 and 6.9 D, respectively. The solvatochromic behavior in 8 and 9 supports the formation of an intramolecular charge transfer (ICT) state. The low fluorescence quantum yields are caused by competitive double bond rotation. The fluorescence decay time of 9 decreases in methyltetrahydrofuran from 2.1 ns at 77 K to 0.11 ns at 200 K. Efficient single bond rotation in 9 was frozen at -50 degrees C in a configuration in which the trimethoxyphenyl ring is perpendicular to the fluorinated rings. 7-9 are photostable compounds. The X-ray structure of 7 shows it is not planar and that its conjugation is distorted. Y-enyne 7 stacks in the solid state showing coulombic, actetylene-arene, and fluorine-pi interactions.

Heterolignanolides: antitumor activity of furyl-, thienyl-, and pyridyl analogs of lignanolides.

A new class of heteroaromatic analogs of lignans, called heterolignanolides, have been tested against several tumor cell lines. These compounds carry diverse heterocyclic rings, instead of the trimethoxyphenyl ring found in the natural lignans yatein and podorhizol. They have moderate antineoplastic activity (P-388, A-549, HT-29) compared with that of yatein. None of the tested compounds has discernible antiviral (HSV-1, VSV) or enzyme inhibitor (ADA, DHFR, GST) activity.

Podophyllotoxin as a probe for the colchicine binding site of tubulin.

The binding of [3H]podophyllotoxin to tubulin, measured by a DEAE-cellulose filter paper method, occurs with an affinity constant of 1.8 X 10(6) M-1 (37 degrees at pH 6.7). Like colchicine, approximately 0.8 mol of podophyllotixin are bound per mol of tubulin dimer, and the reaction is entropy-driven (43 cal deg-1 mol-1). At 37 degrees the association rate constant for podophyllotoxin binding is 3.8 X 10(6) M-1 h-1, approximtaely 10 times higher than for colchicine; this is reflected in the activation energies for binding which are 14.7 kcal/mol for podophyllotoxin and 20.3 kcal/mol for colchicine. The dissociation rate constant for the tubulin-podophyllotoxin complex is 1.9 h-1, and the affinity constant calculated from the ratio of the rates is close to that obtained by equilibrium measurements. Podophyllotxin and colchicine are mutually competitive inhibitors. This can be ascribed to the fact that both compounds have a trimethoxyphenyl ring and analogues of either compound with bulky substituents in their trimethoxyphenyl moiety are unable to inhibit the the binding of either of the two ligands. Tropolone, which inhibits colchicine binding competitively, has no effect on the podophyllotoxin/tubulin reaction. Conversely, podophyllotoxin does not influence tropolone binding. Moreover, the tropolone binding site of tubulin does not show the temperature and pH lability of the colchicine and podophyllotoxin domains, hence this lability can be ascribed to the trimethoxyphenyl binding region of tubulin. Since podophyllotoxin analogues with a modified B ring do not bind, it is concluded that both podophyllotoxin and colchicine each have at least two points of attachment to tubulin and that they share one of them, the binding region of the trimethoxyphenyl moiety.